Flow cytometry is a technology that allows a single cell or particle to be measured for a variety of characteristics, determined by looking at their properties while they flow in a liquid stream. High speed of flow and huge number of objects to be analyzed imposes some strict criteria on which methods can be used for analysis. Most of the known commercial instruments are currently using light scattering for particle sizing and fluorescence detection for chemical recognition. However, vibrational spectroscopy is the only non-invasive optical spectroscopy tool, which has proven to provide chemically-specific information about the interrogated sample. It is hypothesized that vibrational spectroscopy, based on nonlinear Raman scattering can be used to serve as an analytical tool for cytometry by providing rapid and accurate chemical recognition of flowing materials. In the proposed exploratory (R21) research, the idea of ultra-rapid analysis of chemical species will be experimentally tested with an eye on potential accommodation of the developed instrumentation to a commercial flow cytometer. A new instrument will be constructed (Aim 1) to attain the desired parameters needed for rapid, i.e. of the order of 10,000 analyses per second, detection and examination of living cells. The developed instrumentation will be meticulously tested (Aim 2) for its (a) sensitivity, (b) reproducibility, (c) signal- noise-ratio, and (e) speed of spectra acquisition and analysis. Finally (Aim 3), the developed and optimized instrumentation will be tested using a living cell model system to understand the applicability and potential trade-offs of vibrational spectroscopy to flow cytometry of cellular systems. The long-term of this proposal is to develop a rapid, non-invasive methodology for cell and particle analysis, which can be applied for cell/particle analysis and sorting, detection of bacterial pathogens, and cell interactions with pharmaceuticals. The proposed instrument is envisioned to become an important tool in fundamental and clinical biomedical research and will impact many areas of biomedical sciences by providing a novel way to analyze cells and chemical structures at unprecedented speed levels. PUBLIC HEALTH RELEVANCE: Flow cytometry is a technique for counting, examining, and sorting microscopic particles in a stream of fluid. It allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of single cells and particles. A truly non-invasive flow cytometer system, capable of providing chemically specific information, is proposed and will be developed and validated. The proposed technology has applications in a number of fields, such as pathology, immunology, toxicology, and pharmacology.